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Chemical Composition of Membranes
Page 180 5.1— Overview Biological membranes from either eukaryotic or prokaryotic cells have the same classes of chemical components, a similarity in structural organization, and many properties in common. There are major differences in specific lipid, protein, and carbohydrate components but not in physicochemical interaction of these molecules. Membranes have a trilaminar appearance when viewed by electron microscopy (Figure 5.1), with two dark bands on each side of a light band. The overall width of most mammalian membranes is 7–10 nm but some have significantly smaller widths. Intracellular membranes are usually thinner than plasma membranes. Many do not appear symmetrical, with an inner dense layer often thicker than an outer dense layer; there is a chemical asymmetry of membranes. With development of sophisticated techniques for preparation of tissue samples and staining, including negative staining and freeze fracturing, surfaces of membranes have been viewed; at the molecular level surfaces are not smooth but dotted with protruding globularshaped masses. Figure 5.1 Electron micrograph of the erythrocyte plasma membrane showing the trilaminar appearance. A clear space separates the two electrondense lines. Electron microscopy has demonstrated that the inner dense line is frequently thicker than the outer line. Magnification about ×150,000. Courtesy of Dr J. D. Robertson, Duke University, Durham, North Carolina. Membranes are very dynamic structures with a movement that permits cells as well as subcellular structures to adjust their shapes and to change position. Chemical components of membranes, that is, lipids and protein, are ideally suited for their dynamic role. Membranes are an organized sea of lipid in a fluid state, a nonaqueous compartment of cells, in which various components are able to move and interact. Cellular membranes control the composition of space that they enclose by excluding a variety of molecules and by selective transport systems allowing movement of specific molecules from one side to the other. These transporters are proteins. By controlling translocation of substrates, cofactors, ions, and so on, membranes modulate the concentration of substances in cellular compartments, thereby exerting an influence on metabolic pathways. Hormones, and growth and metabolic regulators bind to specific protein receptors on plasma membranes (Chapter 20) and the information to be imparted to the cell is transmitted by the membrane component to the appropriate metabolic pathway by a series of intracellular intermediates, termed second messengers. Plasma membranes of eukaryotic cells also have a role in cellcell recognition, maintenance of the shape of cells, and cell locomotion. The discussion that follows is directed to the chemistry and transport functions of membranes primarily of mammalian cells but the observations and activities described are applicable to all biological membranes. 5.2— Chemical Composition of Membranes Lipids and proteins are the two major components of all membranes. The amount of each varies greatly between different membranes (Figure 5.2). Protein ranges from about 20% in the myelin sheath to over 70% in the inner membrane of the mitochondria. Intracellular membranes have a high percentage of protein because of the large number of enzymic activities of these membranes. Membranes also contain a small amount of various polysaccharides in the form of glycoprotein and glycolipid; there is no free carbohydrate in membranes. Figure 5.2 Representative values for the percentage of lipid and protein in various cellular membranes. Values are for rat liver, except for the myelin and human erythrocyte plasma membrane. Values for liver from other species, including human, indicate a similar pattern. Lipids Are a Major Component of Membranes The three major lipid components of eukaryotic cell membranes are glycerophospholipids, sphingolipids, and cholesterol. Glycerophospholipids and sphingomyelin, a sphingolipid containing phosphate, are classified as phospholipids. Bacteria and bluegreen algae contain glycerolipids where a carbohydrate is attached directly to the glycerol. Individual cellular membranes also contain small quantities of other lipids, such as triacylglycerol and diol derivatives (see the Appendix). Page 181 Glycerophospholipids Are the Most Abundant Lipids of Membranes Glycerophospholipids (phosphoglycerides) have a glycerol molecule as the basic component to which phosphoric acid is esterified at the a carbon (Figure 5.3) and two longchain fatty acids are esterified at the remaining carbon atoms (Figure 5.4). Glycerol does not contain an asymmetric carbon, but the a carbon atoms are not stereochemically identical. Esterification of a phosphate to an a carbon makes the molecule asymmetric. The naturally occurring glycerophospholipids are designated by the stereospecific numbering system (sn)(Figure 5.3) discussed on p. 397. Figure 5.3 Stereochemical con figuration of Lglycerol 3phosphate (snglycerol 3phosphate). The H and OH attached to C2 are above and C1 and C3 are below the plane of the page. 1,2Diacylglycerol 3phosphate, phosphatidic acid, is the parent compound of a series of glycerophospholipids, where different hydroxylcontaining compounds are esterified to the phosphate. The major compounds attached by a phosphodiester bridge to glycerol are choline, ethanolamine, serine, glycerol, and inositol. These structures are presented in Figure 5.5. Phosphatidylethanolamine (ethanolamine glycerophospholipids or the trivial name cephalin) and phosphatidylcholine (choline glycerophospholipid or lecithin) are the most common glycerophospholipids in membranes (Figure 5.6). Phosphatidylglycerol phosphoglyceride (Figure 5.7) (diphosphatidylglycerol or cardiolipin) contains two phosphatidic acids linked by a glycerol and is found nearly exclusively in mitochondrial inner membranes and bacterial membranes. Figure 5.4 Structure of glycerophospholipid. Longchain fatty acids are esterified at C1 and C2 of the Lglycerol 3phosphate. X can be a H (phosphatidic acid) or one of several alcohols presented in Figure 5.5. Figure 5.5 Structures of the major alcohols esterified to phosphatidic acid to form the glycerophospholipid. Page 182 Figure 5.6 Structures of the two most common glycerophospholipids— phosphatidylcholine and phosphatidylethanolamine. Inositol, a hexahydroxy alcohol, is esterified to phosphate in phosphatidylinositol (Figure 5.8). 4Phospho and 4,5bisphosphoinositol glycerophospholipids (Figure 5.8) are present in plasma membranes; the latter is the source of inositol trisphosphate and diacylglycerol that are involved in hormone action (see p. 865). Figure 5.7 Phosphatidylglycerol phosphoglyceride (cardiolipin). Glycerophospholipids contain two fatty acyl groups esterified to carbon atoms 1 and 2 of glycerol; some of the major fatty acids found in glycerophospholipids are presented in Table 5.1. A saturated fatty acid is usually found on C1 of the glycerol and an unsaturated fatty acid on C2. Designation of different glycerophospholipids does not specify which fatty acids are present. Phosphatidylcholine usually contains palmitic or stearic in the sn1 position and a C18 unsaturated fatty acid, oleic, linoleic, or linolenic, on the sn2 carbon. Phosphatidylethanolamine contains palmitic or oleic on sn1 but a longchain polyunsaturated fatty acid, such as arachidonic, on the sn2 position. A saturated fatty acid is a straight chain, as is a fatty acid with an unsaturation in the trans position. A cis double bond, however, creates a kink in the hydrocar Figure 5.8 Phosphatidylinositol. Phosphate groups are also found on C4 or C4 and C5 of the inositol. The additional phosphate groups increase the charge on the polar head of this glycerophospholipid. Page 183 TABLE 5.1 Major Fatty Acids in Glycerophospholipids Common Name Systematic Name Structural Formula Myristic acid nTetradecanoic CH3–(CH2)12–COOH Palmitic acid nHexadecanoic CH3–(CH2)14–COOH Palmitoleic acid cis9Hexadecenoic CH3–(CH2)5–CH=CH–(CH2)7–COOH Stearic acid nOctadecanoic CH3–(CH2)16–COOH Oleic acid cis9Octadecenoic acid CH3–(CH2)7–CH=CH–(CH2)7–COOH Linoleic acid cis,cis9,12Octadecadienoic CH3–(CH2)3–(CH2–CH=CH)2–(CH2)7–COOH Linolenic acid cis,cis,cis9,12,15Octadecatrienoic CH3–(CH2–CH=CH)3–(CH2)7–COOH Arachidonic acid cis,cis,cis,cis5,8,11,14Icosatetraenoic CH3–(CH2)3–(CH2–CH=CH)4–(CH2)3–COOH bon chain (Figure 5.9). A straight chain diagram, as shown in Figures 5.4 and 5.9, does not adequately represent the chemical configuration of a longchain fatty acid. Actually, there is a high degree of coiling of the hydrocarbon chain in a glycerophospholipid that is disrupted by a double bond. The presence of unsaturated fatty acids has a marked effect on the physicochemical state of the membrane (see p. 195). Glycerol ether phospholipids contain a long aliphatic chain in ether linkage to the glycerol at the sn1 position (Figure 5.10). Ether phospholipids contain an alkyl group (alkyl acylglycerophospholipid) or an a , b unsaturated ether, termed a plasmalogen. The latter groups are more prevalent in membranes. Plasmalogens containing ethanolamine (ethanolamine plasmalogen) and choline (choline plasmalogen) esterified to the phosphate are abundant in nervous tissue and heart but not in liver. In human hearts more than 50% of the ethanolamine glycerophospholipids are plasmalogens. Glycerophospholipids are amphipathic, containing both a polar end, or head group, due to the charged phosphate and substitutions on the phosphate, and a nonpolar tail due to hydrophobic hydrocarbon chains of the fatty acyl Figure 5.9 Conformation of fatty acyl groups in phospholipids. The saturated and unsaturated fatty acids with trans double bonds are straight chains in their minimum energy conformation, whereas a chain with a cis double bond has a bend. The trans double bond is rare in naturally occurring fatty acids. Figure 5.10 Ethanolamine plasmalogen. Note the ether linkage of the aliphatic chain on C1 of glycerol. Page 184 TABLE 5.2 Predominant Charge on Glycerophospholipids and Sphingomyelin at pH 7.0 Lipid Phosphate Group Base Net Charge Phosphatidylcholine –1 +1 0 Phosphatidylethanolamine –1 +1 0 Phosphatidylserine –1 +1, –1 –1 Phosphatidylglycerol –1 0 –1 Diphosphatidylglycerol (cardiolipin) –2 0 –2 Phosphatidylinositol –1 0 –1 Sphingomyelin –1 +1 0 groups. The polar groups are charged at pH 7.0 with a negative charge due to ionization of the phosphate group (pK 2) and charges from groups esterified to phosphate (Table 5.2). Choline and ethanolamine glycerophospholipids are zwitterions at pH 7.0, with both a negative charge from phosphate and a positive charge on nitrogen. Phosphatidylserine has two negative charges, one on phosphate and one on the carboxyl group of serine, and a positive charge on the a amino group of serine, with a net charge of –1 at pH 7.0. In contrast, glycerophospholipids containing inositol and glycerol have only a single negative charge on phosphate; 4 phospho and 4,5bisphosphoinositol derivatives are very polar compounds with additional negative charges on the phosphate groups. Every tissue and cellular membrane has a distinctive composition of glycerophospholipids and a definite pattern in fatty acid composition. There is a greater variability in the fatty acyl groups of different tissues in a single species than in the fatty acyl groups of the same tissue in a variety of species. In addition, the fatty acid content of the glycerophospholipids can vary, depending on the physiological or pathophysiological state of the tissue. Figure 5.11 Structures of sphingosine and dihydrosphingosine. Sphingolipids Are Also Present in Membranes The amino alcohols sphingosine (D4sphingenine) and dihydrosphingosine (Figure 5.11) are the basis for another series of membrane lipids, the sphingolipids. A ceramide is sphingosine with a saturated or unsaturated longchain fatty acyl group in amide linkage on the amino group (Figure 5.12). With two nonpolar tails a ceramide is similar in structure to diacylglycerol. Various substitutions are found on the hydroxyl group at position 1. The sphingomyelin series has phosphorylcholine esterified to the 1OH (Figure 5.13) and is the most abundant sphingolipid in mammalian tissues. The similarity of this structure to choline glycerophospholipids is apparent, and they have many properties in common; note that the sphingomyelins are amphipathic compounds with a charged head group. Sphingomyelins and glycerophospholipids are classified as phospholipids. The sphingomyelin of myelin contains predominantly the longer chain fatty acids, with carbon lengths of 24; as with glycerophospholipids, there is a specific fatty acid composition of the sphingomyelin, depending on the tissue. Figure 5.12 Structure of a ceramide. Glycosphingolipids do not contain phosphate and have a sugar attached by a b glycosidic linkage to the 1OH group of the sphingosine in a ceramide. One subgroup is the cerebrosides, which contain either a glucose (glucocerebrosides) or galactose (galactocerebrosides) attached to a ceramide (Figure 5.14). Cerebrosides are neutral compounds. Galactocerebrosides are found predominantly in brain and nervous tissue, whereas the small quantities of cerebrosides in nonneural tissues usually contain glucose. Phrenosin, a specific galac Page 185 tocerebroside, contains a 2OH C24 fatty acid. Galactocerebrosides may contain a sulfate group esterified on the 3 position of the sugar. They are called sulfatides (Figure 5.15). Cerebrosides and sulfatides usually contain fatty acids with 22–26 carbon atoms. In place of monosaccharides, neutral glycosphingolipids often have 2 (dihexosides), 3 (trihexosides), or 4 (tetrahexosides) sugar residues attached to the 1OH group of sphingosine. Diglucose, digalactose, Nacetylglucosamine, and Nacetyldigalactosamine are the usual sugars. The most complex group of glycosphingolipids, the gangliosides, contain oligosaccharide head groups with one or more residues of sialic acid; these are amphipathic compounds with a negative charge at pH 7.0. The gangliosides represent 5–8% of the total lipids in brain, and some 20 different types have been identified differing in the number and relative position of the hexose and sialic acid residues. This is the basis of their classification; a detailed description of the nomenclature and structures of gangliosides is presented on p. 426. Figure 5.13 Structure of a choline containing sphingomyelin. Figure 5.14 Structure of a galactocere broside containing a C24 fatty acid. Figure 5.15 Structure of a sulfatide. Page 186 Figure 5.16 Structure of cholesterol. Most Membranes Contain Cholesterol Cholesterol is the third major lipid in membranes. With four fused rings and an eightmember branched hydrocarbon chain attached to the D ring at position 17, cholesterol is a compact, rigid, hydrophobic molecule (Figure 5.16). It also has a polar hydroxyl group at C3. Lipid Composition Varies in Different Membranes There are quantitative differences between the classes of lipids and individual lipids in various cell membranes (Figure 5.17). The lipid composition is very similar in the same intracellular membrane of a specific tissue in different species. The plasma membrane exhibits the greatest variation in percentage composition because the amount of cholesterol is affected by the nutritional state of the animal. Plasma membranes have the highest concentration of neutral lipids and sphingolipids; myelin membranes of axons of neural tissue are rich in sphingolipids, with a high proportion of glycosphingolipids. Intracellular membranes primarily contain glycerophospholipids with little sphingolipids or cholesterol. The membrane lipid composition of mitochondria, nuclei, and rough endoplasmic reticulum are similar, with Golgi membrane being somewhere between other intracellular membranes and the plasma membrane. As indicated previously, cardiolipin is found nearly exclusively in the inner mitochondrial membrane. Choline containing lipids, phosphatidylcholine, and sphingomyelin, are predominant, with ethanolamine glycerophospholipid second. The constancy of composition of various membranes indicates the relationship between lipids and the specific functions of individual membranes. Membrane Proteins Are Classified Based on Their Ease of Removal Membrane proteins are classified on the basis of ease of removal from isolated membrane fractions. Peripheral (or extrinsic) proteins are released from a Figure 5.17 Lipid composition of cellular membranes isolated from rat liver. (a) Amount of major lipid components as percentage of total lipid. The area labeled ''Other" includes mono, di, and triacylglycerol, fatty acids, and cholesterol esters. (b) Phospholipid composition as a percentage of total phospholipid. Values from R. Harrison and G. G. Lunt, Biological Membranes. New York Wiley, 1975.